- Email: [email protected]
Genetic polymorphisms of the sortase A gene and early childhood caries in two-year-old children
archives of oral biology 57 (2012) 948–953
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Genetic polymorphisms of the sortase A gene and early childhood caries in two-year-old children X.H. Zhang, Y. Zhou, Q.H. Zhi, Y. Tao, H.C. Lin * Department of Preventive Dentistry, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, China
article info
abstract
Article history:
Objective: To explore and compare the genetic polymorphisms of the sortase A (srtA) gene
Accepted 1 February 2012
found in Streptococcus mutans (S. mutans) infecting two-year-old children suffering early childhood caries to those found in caries-free children through molecular identification
Keywords:
methods.
Streptococcus mutans
Methods: Clinical S. mutans strains were isolated from the dental plaques of two-year-old
Sortase
children. Fifteen strains of S. mutans from the caries-active group and 15 strains of S. mutans
Caries
from the caries-free group were collected. Genomic DNA was extracted from the S. mutans isolates. DNA fragments, including the srtA gene, were amplified by PCR. The PCR products were purified, sequenced and analyzed. A chi-square test and BioEdit software were used to analyze the sequencing results. Results: All 30 clinically isolated S. mutans strains had a 741 base pair (bp) srtA gene. There were no nucleotide sequence insertions or deletions observed in the srtA genes. Twenty mutations were identified in the srtA genes that taken from the 30 clinical strains. There were 10 silent point mutations at the 78, 99, 150, 165, 186, 222, 249, 261, 312, and 636 bp positions. The other 10 mutations were point mutations resulting in a missense mutation at the 23, 34, 36, 47, 112, 114, 168, 176, 470, and 671 bp positions. None of the positions were enzyme-activity sites of srt A. The missense mutation rates of the two groups did not exhibit statistically significant differences. Conclusion: There were no genetic polymorphisms of the sortase A gene associated with early childhood caries in two-year-old children. # 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Streptococcus mutans (S. mutans) is considered a principal etiological agent of human dental caries.1 The main virulence factors associated with cariogenicity include adhesion, acidogenicity, and acid tolerance.2 The adherence of S. mutans to dental surfaces is the first step in the formation of a plaque biofilm and is mediated by both sucrose-dependent and sucrose-independent mechanisms.3,4 Incipient adherence of
S. mutans is a sucrose-independent mechanism. In the absence of sucrose, S. mutans expresses several surface proteins that can bind to salivary components to form the required pellicle on the teeth. This sucrose-independent adhesion is implicated in initial S. mutans colonization of the teeth in vivo. Recently, it was demonstrated that the sortase A (SrtA) enzyme is responsible for sorting and anchoring surface proteins to the cell wall of S. mutans.5 SrtA is a transpeptidase essential for anchoring the majority of the LPXTG motifcontaining proteins of gram-positive bacteria,6 and inactiva-
* Corresponding author at: Preventive Dentistry Department, Guanghua School of Stomatology, Ling Yuan Road West 56, Guangzhou 510055, China. Tel.: +86 20 83860175; fax: +86 20 83822807. E-mail address: [email protected] (H.C. Lin). 0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2012.02.002
archives of oral biology 57 (2012) 948–953
tion of the srtA gene in several gram-positive bacteria has been reported to cause multiple pathogenesis defects.7,8 S. mutans strains with a mutation in srtA that disables the enzyme are almost incapable of colonizing the teeth in the absence of sucrose,9 and srtA is responsible for the cariogenicity of S. mutans.5 Early childhood caries (ECC) provides one of the major disease impacts on children’s health. Early Childhood Caries is defined as ‘‘the presence of 1 or more decayed (noncavitated or cavitated lesions), missing (due to caries), or filled tooth surfaces’’ in any primary tooth in a 71-month or younger child.10 ECC develops early and is distributed in a polarized pattern with most dental decay occurring in a small number of children. There are many reports that S. mutans has a strong relationship with ECC.11,12 It has been proposed that the strains of S. mutans associated with ECC are genetically distinct from those found in caries-free (CF) children.13 As srtA is important in the control of surface proteins during the initial S. mutans colonization of the teeth, we hypothesized that the srtA gene might possess genetic polymorphisms in different strains of S. mutans. The purpose of this study was to explore and compare the genetic polymorphisms of the srtA gene found in S. mutans associated with ECC to those found in S. mutans in CF children through molecular identification methods in clinical isolates obtained from two-year-old children.
949
stationary phase. The cells were harvested by centrifugation at 12,000 rpm for 5 min and washed twice with phosphate buffered saline. The bacterial cells were suspended in 5% Chelex 100, a chelating exchange resin,17 treated with 5 units (20 mg/ml) proteinase K at 37 8C for 1 min, then digested at 56 8C for 4 h followed by boiling for 10 min and immediate cooling on ice for 3 min. After centrifugation at 12,000 rpm for 3 min, the supernatant was collected as a PCR template. The DNA concentration and purity were determined spectrophotometrically by measuring the A260 and A280 (Varian, USA). The genomic DNA samples were stored at 80 8C before use. Genomic DNA from S. mutans UA159 was used as the reference.
2.3.
Amplification of srtA gene
The PCR primers were designed according to the UA159 srtA sequence. The srtA gene was localized in the 1053014– 1053754 bp position within the UA159 whole genome, and the total length of srtA was 741 bp. Primer Express 2.0 software was used for designing the primers. The designed primer was used to amplify a 941 bp DNA fragment carrying the srtA gene. The primers were synthesized using an ABI 3900 DNA synthesizer. Forward primer: 50 -TTGTTATTACGTTTGCAATGCTCA-30 Reverse primer: 50 -CAAAGTGATGCGCCTAGATGAA-30
2.
Materials and methods
2.1.
Bacterial strains
All clinical isolates were preserved from our previous study.14 Fifteen strains of S. mutans were isolated separately from caries-active (dmft 5) children and caries-free (dmft = 0) children. The dmft index is the number of decayed, missing and filled deciduous teeth. The children were two-year-old who participated in an epidemiological survey. This survey included 394 children, with 109 having ECC and 285 being CF.15 Clinical examinations for ECC were conducted with the aid of CPI probes, disposable mouth mirrors, and an intra-oral LED light source using the criteria recommended by World Health Organization.16 We selected 32 caries-active (dmft 5) children from 109 children with ECC and randomly selected 32 CF (dmft = 0) children from 285 CF children matched with sex. Dental plaque samples from all 64 children were cultured and S. mutans colonies were isolated only from 24 children in the caries-active group and 12 children in the CF group. Then, we randomly picked 2–4 colonies according to morphology from each child for gram staining and biochemical tests and got 75 isolated S. mutans strains from caries-active group and 34 strains from the CF group. 15 strains from each of the two groups were randomly selected as research objects. The S. mutans strains were grown in brain heart infusion broth under anaerobic conditions (10%H2, 10% CO2, 80% N2) in anaerobic jars. S. mutans UA159 was used as a reference strain.
2.2.
DNA extraction
The clinical isolates were transferred to 2 ml of brain heart infusion broth and incubated overnight at 37 8C until reaching
The PCR amplification was performed in a 50 ml total reaction volume. Using a hot start protocol, the samples were preheated at 93 8C for 3 min followed by amplification under the following conditions: denaturation at 93 8C for 45 s, annealing at 55 8C for 45 s, and elongation at 72 8C for 45 s. A total of 40 cycles were performed and followed by a final elongation step at 72 8C for 45 s. Eight microliters of each amplified product was electrophoresed in 2% (wt/vol) agarose gel along with a molecular size marker (Takara, Japan) in parallel. The Tris–borate–EDTA buffer electrophoresis was performed at 110 V for 0.5 h. The gel was stained with ethidium bromide and visualized under shortwavelength UV light. The results were captured with an Alpha IS-1000 digital imaging system (Alpha Innotech Corp., San Leandro, CA).
2.4.
Sequencing of srtA gene
The PCR products were collected and purified using a Qiaquick Gel Extraction Kit (QIAgen, Hilden, Germany). Each amplified product was sequenced at both directions by the Shanghai Shenggong Bioengineering Company (Shenggong, China). The sequences of the PCR products were compared with known srtA gene sequences in GenBank from the UA159 strain using BioEdit software (GenBank accession number NC_004350).
2.5.
Statistical analysis
SPSS17.0 software was used for statistical analysis. A chisquare test was used to compare different srtA sequences between the caries-active group and the caries-free group. A Pvalue below 0.05 was used to indicate a statistically significant
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archives of oral biology 57 (2012) 948–953
difference between the two groups. BioEdit software was used to analyze the results of the sequencing.
3.
Results
3.1.
PCR products
A 941 bp DNA fragment carrying the srtA gene was amplified from the chromosome of all clinical bacterial strains by PCR. All 30 strains demonstrated clear banding patterns and presented a single positive band of 900–1000 bp in length.
3.2.
Sequencing results
1. The 741 bp srtA genes from the 30 clinical strains were sequenced. None of the srtA genes in the sequences trains had a base insertion or deletion.
2. Point mutations were present in the sequences of one of the clinical isolates (Fig. 1). In the caries-active group, 12 strains had point mutations, and the remaining three strains had no mutations for a total mutation percentage of 80%. In the caries-free group, all of the strains had point mutations for a total mutation percentage of 100%. There was no significance difference between the two groups (chi-square test, P = 0.224). 3. The sequencing revealed 20 mutation loci among the 30 clinical isolates. Half of the mutations were silent mutations. The remaining mutations were missense mutations. The transversion of the amino acids according to codon were shown in Table 1. 4. The silent mutation loci of the clinical isolates included position 78, 99, 150, 165, 186, 222, 249, 261, 312, and 636. The mutation loci details were listed in Table 2. 5. The missense mutation loci of the clinical isolates included positions 23, 34, 36, 47, 112, 114, 168, 176, 470, and 671. The mutation loci details are listed in Table 3. The mutation loci of 23, 34, 36, 47, and 470 were only noted in the caries-active group. Locus 168 was only noted in the caries-free group.
Fig. 1 – Point mutation in a clinical isolate (number is caries active 1), as indicated by srtA sequence. We can saw the point mutations occurred in the 78, 99, 112, 114, 222, 249, 470 locus base in the caries active 1 clinical isolate.
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archives of oral biology 57 (2012) 948–953
Table 1 – Transversion of amino acid according to codons. Mutation
Base site
UA159
Clinical isolates
Codon
Amino acid
Codon
Amino acid
Silent mutation
78 99 150 165 186 222 249 261 312 636
GGT ACT TCT AAA GCT TCC GCA GCT AAC GCC
Glycin Threonine Serine Lysine Alanine Serine Alanine Alanine Asparagine Alanine
GGC ACC TCC AAG GCG TCT GCG GCC AAT GCT
Glycin Threonine Serine Lysine Alanine Serine Alanine Alanine Asparagine Alanine
Missense mutation
23 34 36 47 112 114 168 176 470 671
AGG AGT AGT ACC GCC GCC GAT CAC CGT AAT
Arginine Serine Serine Threonine Alanine Alanine Asparagine Histidine Arginine Asparagine
AAG GGC GGC ATC ACT ACT GAG CGC CAT ACT
Lysine Glycin Glycin Isoleucine Threonine Threonine Glutamic acid Arginine Histidine Threonine
Table 2 – Details of silent mutation sites in carie-active group and caries-free group. Clinical isolates
CA1a CA2 CA3 CA4 CA5 CA6 CA7 CA8 CA9 CA10 CA11 CA12 CA13 CA14 CA15 CF1c CF2 CF3 CF4 CF5 CF6 CF7 CF8 CF9 CF10 CF11 CF12 CF13 CF14 CF15 a b c
Base loci 78
99
T!Cb T!C T!C T!C T!C T!C
T!C T!C T!C T!C T!C T!C
T!C T!C
T!C
T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C
T!C T!C T!C T!C T!C T!C
150
186
A!G A!G
T!C T!C T!C T!C T!C T!C
A!G A!G A!G A!G A!G A!G
T!C T!C T!C T!C T!C T!C T!C
A!G A!G A!G
T!C T!C T!C T!C T!C T!C
165
T!C
A!G A!G A!G A!G A!G A!G A!G
222
249
C!T C!T C!T C!T C!T C!T
A!G A!G A!G A!G A!G A!G
261
312
636
C!T C!T C!T C!T C!T
T!C T!C T!C T!C T!C T!C
T!G T!G T!G
T!C T!C T!C C!T C!T C!T C!T C!T C!T
A!G A!G A!G A!G A!G A!G
C!T C!T C!T C!T C!T C!T
C!T C!T C!T C!T
T!C T!C T!C C!T C!T C!T
A!G A!G A!G
CA, caries-active group. T!C, T represents the 78 locus base in UA159, C represents the 78 locus base in the clinical isolate. CF, caries-free group.
C!T C!T C!T
C!T
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archives of oral biology 57 (2012) 948–953
Table 3 – Details of missense mutation sites in carie-active group and caries-free group. Clinical isolates
Base loci 23
34
36
47
a
CA1 CA2 CA3 CA4 CA5 CA6 CA7 CA8 CA9 CA10 CA11 CA12 CA13 CA14 CA15 CF1c CF2 CF3 CF4 CF5 CF6 CF7 CF8 CF9 CF10 CF11 CF12 CF13 CF14 CF15 Chi-square test a b c d
4.
112 G!A G!A G!A G!A G!A G!A
G!A G!A
A!G A!G
T!C T!C
C!T C!T
G!A
A!G
T!C
C!T
b
114
168
176
C!T C!T C!T C!T C!T C!T
470 G!A
A!C A!C A!C A!C A!C
A!G A!G A!G A!G A!G A!G
G!A
A!G A!G A!G
d
P = 0.224
P = 0.224
P = 0.224
P = 0.224
G!A G!A G!A G!A G!A G!A
C!T C!T C!T C!T C!T C!T
G!A G!A G!A P = 0.466
C!T C!T C!T P = 0.466
671
T!G T!G
T!G T!G
A!G A!G A!G A!G A!G A!G A!G
P = 0.100
P = 0.272
P = 0.483
A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C P = 0.100
CA, caries-active group. G!A, G represents the 112 locus base in UA159, A represents the 112 locus base in the clinical isolates. CF, caries-free group. There is no significant statistical difference between the two groups regarding the mutation rate at each locus (P > 0.05).
Discussion
It has long been known that considerable phenotypic variation exists within S. mutans strains.18 This variation can be a consequence of a variety of different genetic events, including local point mutations, translocations and inversions. The availability of the full genome sequence of S. mutans UA159 has allowed comparison between strains at the genome level. The PCR-based double sequencing approach identifies single point mutations. Though restriction fragment length polymorphism (RFLP) is a classic method of biosystems gene structure analysis, it is not easy to select the proper restriction enzymes and the precise restriction enzyme cutting sites. In comparison to RFLP, the PCR-based double sequencing approach is considerably more objective in indicating the real genomic variations that may not always be visible via the electrophoretic band patterns in RFLP. Sortase has been shown to be a protease that catalyzes the cell wall anchoring of surface proteins containing an LPXTG motif in gram-positive bacteria. The srtA gene is required for cell wall anchoring and the surface display of antigen I/II (P1),5 glucan binding protein C (GbpC),19 dextranase,20 and surface proteins with LPXTG motif sorting signals. The inactivation of
srtA causes a decrease in biofilm formation and colonization.8,21 SrtA plays an important role in modulating the cariogenicity of S. mutans.5 The results of the present study indicated that clinical isolates of S. mutans had full-length srtA genes when compared to S. mutans UA159. All clinical isolates possessed point mutations. These results suggest those clinical strains have undergone genetic evolution. The two study groups have 20 mutation sites overall with both missense mutations and silent mutations represented. A missense mutation is a point mutation in which a single nucleotide change results in a codon that code for a different amino acid. A silent mutation is a DNA mutation that does not result in a change to the expressed amino acid. The structure of sortase reveals that the absolutely conserved His120, Cys184, and Arg197 residues of sortases reside within the active site.22 The 20 mutation sites identified in the present study were not in the sortase enzyme’s active sites. This finding means that the point mutations of the clinical strains did not affect the activity of the sortase enzyme. The missense mutation rate between the two groups was not significantly different. This probably reflects that the function of the srtA gene in clinical isolates is highly
archives of oral biology 57 (2012) 948–953
conserved. Similar results have been reported in other studies.23,24 On the other hand, the sample was relatively small for difference analysis between the two groups. The dmft 5 set as cut-off value probably narrow the selection objects of caries-active group. These two points may have some importance in the lack of significance. However, the results of the present study still provide some clues for the study of srtA polymorphisms in the future. The sequences were compared to UA159, which is a cariogenic strain of S. mutans. There were mutational sites observed in the cariesactive group that had no effect on cariogenicity, and mutation sites were observed in the caries-free group that modified cariogenicity. These sites needs further study. There was no significant difference in srtA enzyme activity between the two groups. These results indicate that the genetic diversity within the S. mutans srtA gene is not related to the severe caries experience in deciduous dentition. Further studies are needed to explore more fully the relationship between srtA and ECC.
5.
Conclusions
The results of the present study suggest that the genetic diversity within the srtA gene of S. mutans is not related to severe caries experiences in deciduous dentition. There were no detected genetic polymorphisms of the srtA gene in S. mutans isolates taken from two-year-old children suffering early childhood caries.
Acknowledgments Funding: This work was supported by the Guangdong Provincial Science & Technology project (Grant No. 2007B060401026). Competing interests: The authors declare that there are no conflicts of interests. Ethical approval: Ethical Approval has been given by the Research Ethics Committee of Guanghua School of Stomatology, Sun Yat-sen University (No: 2009-01).
references
1. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986;50(4):353–80. 2. Banas JA. Virulence properties of Streptococcus mutans. Front Biosci 2004;9:1267–77. 3. Cvitkovitch DG, Li YH, Ellen RP. Quorum sensing and biofilm formation in Streptococcal infections. J Clin Invest 2003;112(11):1626–32. 4. Koga T, Asakawa H, Okahashi N, Hamada S. Sucrosedependent cell adherence and cariogenicity of serotype c Streptococcus mutans. J Gen Microbiol 1986;132(10): 2873–83. 5. Lee SF, Boran TL. Roles of sortase in surface expression of the major protein adhesin P1, saliva-induced aggregation and adherence, and cariogenicity of Streptococcus mutans. Infect Immun 2003;71(2):676–81.
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6. Dramsi S, Trieu-Cuot P, Bierne H. Sorting sortases: a nomenclature proposal for the various sortases of Grampositive bacteria. Res Microbiol 2005;156(3):289–97. 7. Bierne H, Mazmanian SK, Trost M, Pucciarelli MG, Liu G, Dehoux P, et al. Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence. Mol Microbiol 2002;43(4):869–81. 8. Yamaguchi M, Terao Y, Ogawa T, Takahashi T, Hamada S, Kawabata S. Role of Streptococcus sanguinis sortase A in bacterial colonization. Microbes Infect 2006;8(12–13):2791–6. 9. Igarashi T. Deletion in sortase gene of Streptococcus mutans Ingbritt. Oral Microbiol Immunol 2004;19(3):210–3. 10. Drury TF, Horowitz AM, Ismail AI, Maertens MP, Rozier RG, Selwitz RH. Diagnosing and reporting early childhood caries for research purposes. A report of a workshop sponsored by the National Institute of Dental and Craniofacial Research, the Health Resources and Services Administration, and the Health Care Financing Administration. J Public Health Dent 1999;59(3):192–7. 11. Corby PM, Lyons-Weiler J, Bretz WA, Hart TC, Aas JA, Boumenna T, et al. Microbial risk indicators of early childhood caries. J Clin Microbiol 2005;43(11):5753–9. 12. Kanasi E, Johansson I, Lu SC, Kressin NR, Nunn ME, Kent Jr R et al. Microbial risk markers for childhood caries in pediatricians’ offices. J Dent Res 2010;89(4):378–83. 13. Saxena D, Li Y, Caufield PW. Identification of unique bacterial gene segments from Streptococcus mutans with potential relevance to dental caries by subtraction DNA hybridization. J Clin Microbiol 2005;43(7):3508–11. 14. Zhang XH, Zhou Y, Lin HC. Acquisition of Streptococcus mutans and cell adherence among 2-year old children with different caries experience. Chinese Journal of Practical Stomatology 2009;2(12):727–9. [in Chinese]. 15. Zhou Y, Lin HC, Lo EC, Wong MC. Risk indicators for early childhood caries in 2-year-old children in southern China. Aust Dent J 2011;56(1):33–9. 16. World Health Organization. Oral health survey: basic methods. 4th ed. Geneva: World Health Organization; 1997. p. 39–46. 17. de Lamballerie X, Zandotti C, Vignoli C, Bollet C, de Micco P. A one-step microbial DNA extraction method using ‘‘Chelex 100’’ suitable for gene amplification. Res Microbiol 1992;143(8):785–90. 18. Waterhouse JC, Swan DC, Russell RR. Comparative genome hybridization of Streptococcus mutans strains. Oral Microbiol Immunol 2007;22(2):103–10. 19. Igarashi T, Asaga E, Sato Y, Goto N. Inactivation of srtA gene of Streptococcus mutans inhibits dextran-dependent aggregation by glucan-binding protein C. Oral Microbiol Immunol 2004;19(1):57–60. 20. Igarashi T, Asaga E, Goto N. Roles of Streptococcus mutans dextranase anchored to the cell wall by sortase. Oral Microbiol Immunol 2004;19(2):102–5. 21. Le´vesque CM, Voronejskaia E, Huang YC, Mair RW, Ellen RP, Cvitkovitch DG. Involvement of sortase anchoring of cell wall proteins in biofilm formation by Streptococcus mutans. Infect Immun 2005;73(6):3773–7. 22. Ilangovan U, Ton-That H, Iwahara J, Schneewind O, Clubb RT. Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus. Proc Natl Acad Sci USA 2001;98(11):6056–61. 23. Paterson GK, Mitchell TJ. The role of Streptococcus pneumoniae sortase A in colonisation and pathogenesis. Microbes Infect 2006;8(1):145–53. 24. Osaki M, Takamatsu D, Shimoji Y, Sekizaki T. Allelic variation in srtAs of Streptococcus suis strains. FEMS Microbiol Lett 2003;219(2):195–201.
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Genetic polymorphisms of the sortase A gene and early childhood caries in two-year-old children X.H. Zhang, Y. Zhou, Q.H. Zhi, Y. Tao, H.C. Lin * Department of Preventive Dentistry, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, China
article info
abstract
Article history:
Objective: To explore and compare the genetic polymorphisms of the sortase A (srtA) gene
Accepted 1 February 2012
found in Streptococcus mutans (S. mutans) infecting two-year-old children suffering early childhood caries to those found in caries-free children through molecular identification
Keywords:
methods.
Streptococcus mutans
Methods: Clinical S. mutans strains were isolated from the dental plaques of two-year-old
Sortase
children. Fifteen strains of S. mutans from the caries-active group and 15 strains of S. mutans
Caries
from the caries-free group were collected. Genomic DNA was extracted from the S. mutans isolates. DNA fragments, including the srtA gene, were amplified by PCR. The PCR products were purified, sequenced and analyzed. A chi-square test and BioEdit software were used to analyze the sequencing results. Results: All 30 clinically isolated S. mutans strains had a 741 base pair (bp) srtA gene. There were no nucleotide sequence insertions or deletions observed in the srtA genes. Twenty mutations were identified in the srtA genes that taken from the 30 clinical strains. There were 10 silent point mutations at the 78, 99, 150, 165, 186, 222, 249, 261, 312, and 636 bp positions. The other 10 mutations were point mutations resulting in a missense mutation at the 23, 34, 36, 47, 112, 114, 168, 176, 470, and 671 bp positions. None of the positions were enzyme-activity sites of srt A. The missense mutation rates of the two groups did not exhibit statistically significant differences. Conclusion: There were no genetic polymorphisms of the sortase A gene associated with early childhood caries in two-year-old children. # 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Streptococcus mutans (S. mutans) is considered a principal etiological agent of human dental caries.1 The main virulence factors associated with cariogenicity include adhesion, acidogenicity, and acid tolerance.2 The adherence of S. mutans to dental surfaces is the first step in the formation of a plaque biofilm and is mediated by both sucrose-dependent and sucrose-independent mechanisms.3,4 Incipient adherence of
S. mutans is a sucrose-independent mechanism. In the absence of sucrose, S. mutans expresses several surface proteins that can bind to salivary components to form the required pellicle on the teeth. This sucrose-independent adhesion is implicated in initial S. mutans colonization of the teeth in vivo. Recently, it was demonstrated that the sortase A (SrtA) enzyme is responsible for sorting and anchoring surface proteins to the cell wall of S. mutans.5 SrtA is a transpeptidase essential for anchoring the majority of the LPXTG motifcontaining proteins of gram-positive bacteria,6 and inactiva-
* Corresponding author at: Preventive Dentistry Department, Guanghua School of Stomatology, Ling Yuan Road West 56, Guangzhou 510055, China. Tel.: +86 20 83860175; fax: +86 20 83822807. E-mail address: [email protected] (H.C. Lin). 0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2012.02.002
archives of oral biology 57 (2012) 948–953
tion of the srtA gene in several gram-positive bacteria has been reported to cause multiple pathogenesis defects.7,8 S. mutans strains with a mutation in srtA that disables the enzyme are almost incapable of colonizing the teeth in the absence of sucrose,9 and srtA is responsible for the cariogenicity of S. mutans.5 Early childhood caries (ECC) provides one of the major disease impacts on children’s health. Early Childhood Caries is defined as ‘‘the presence of 1 or more decayed (noncavitated or cavitated lesions), missing (due to caries), or filled tooth surfaces’’ in any primary tooth in a 71-month or younger child.10 ECC develops early and is distributed in a polarized pattern with most dental decay occurring in a small number of children. There are many reports that S. mutans has a strong relationship with ECC.11,12 It has been proposed that the strains of S. mutans associated with ECC are genetically distinct from those found in caries-free (CF) children.13 As srtA is important in the control of surface proteins during the initial S. mutans colonization of the teeth, we hypothesized that the srtA gene might possess genetic polymorphisms in different strains of S. mutans. The purpose of this study was to explore and compare the genetic polymorphisms of the srtA gene found in S. mutans associated with ECC to those found in S. mutans in CF children through molecular identification methods in clinical isolates obtained from two-year-old children.
949
stationary phase. The cells were harvested by centrifugation at 12,000 rpm for 5 min and washed twice with phosphate buffered saline. The bacterial cells were suspended in 5% Chelex 100, a chelating exchange resin,17 treated with 5 units (20 mg/ml) proteinase K at 37 8C for 1 min, then digested at 56 8C for 4 h followed by boiling for 10 min and immediate cooling on ice for 3 min. After centrifugation at 12,000 rpm for 3 min, the supernatant was collected as a PCR template. The DNA concentration and purity were determined spectrophotometrically by measuring the A260 and A280 (Varian, USA). The genomic DNA samples were stored at 80 8C before use. Genomic DNA from S. mutans UA159 was used as the reference.
2.3.
Amplification of srtA gene
The PCR primers were designed according to the UA159 srtA sequence. The srtA gene was localized in the 1053014– 1053754 bp position within the UA159 whole genome, and the total length of srtA was 741 bp. Primer Express 2.0 software was used for designing the primers. The designed primer was used to amplify a 941 bp DNA fragment carrying the srtA gene. The primers were synthesized using an ABI 3900 DNA synthesizer. Forward primer: 50 -TTGTTATTACGTTTGCAATGCTCA-30 Reverse primer: 50 -CAAAGTGATGCGCCTAGATGAA-30
2.
Materials and methods
2.1.
Bacterial strains
All clinical isolates were preserved from our previous study.14 Fifteen strains of S. mutans were isolated separately from caries-active (dmft 5) children and caries-free (dmft = 0) children. The dmft index is the number of decayed, missing and filled deciduous teeth. The children were two-year-old who participated in an epidemiological survey. This survey included 394 children, with 109 having ECC and 285 being CF.15 Clinical examinations for ECC were conducted with the aid of CPI probes, disposable mouth mirrors, and an intra-oral LED light source using the criteria recommended by World Health Organization.16 We selected 32 caries-active (dmft 5) children from 109 children with ECC and randomly selected 32 CF (dmft = 0) children from 285 CF children matched with sex. Dental plaque samples from all 64 children were cultured and S. mutans colonies were isolated only from 24 children in the caries-active group and 12 children in the CF group. Then, we randomly picked 2–4 colonies according to morphology from each child for gram staining and biochemical tests and got 75 isolated S. mutans strains from caries-active group and 34 strains from the CF group. 15 strains from each of the two groups were randomly selected as research objects. The S. mutans strains were grown in brain heart infusion broth under anaerobic conditions (10%H2, 10% CO2, 80% N2) in anaerobic jars. S. mutans UA159 was used as a reference strain.
2.2.
DNA extraction
The clinical isolates were transferred to 2 ml of brain heart infusion broth and incubated overnight at 37 8C until reaching
The PCR amplification was performed in a 50 ml total reaction volume. Using a hot start protocol, the samples were preheated at 93 8C for 3 min followed by amplification under the following conditions: denaturation at 93 8C for 45 s, annealing at 55 8C for 45 s, and elongation at 72 8C for 45 s. A total of 40 cycles were performed and followed by a final elongation step at 72 8C for 45 s. Eight microliters of each amplified product was electrophoresed in 2% (wt/vol) agarose gel along with a molecular size marker (Takara, Japan) in parallel. The Tris–borate–EDTA buffer electrophoresis was performed at 110 V for 0.5 h. The gel was stained with ethidium bromide and visualized under shortwavelength UV light. The results were captured with an Alpha IS-1000 digital imaging system (Alpha Innotech Corp., San Leandro, CA).
2.4.
Sequencing of srtA gene
The PCR products were collected and purified using a Qiaquick Gel Extraction Kit (QIAgen, Hilden, Germany). Each amplified product was sequenced at both directions by the Shanghai Shenggong Bioengineering Company (Shenggong, China). The sequences of the PCR products were compared with known srtA gene sequences in GenBank from the UA159 strain using BioEdit software (GenBank accession number NC_004350).
2.5.
Statistical analysis
SPSS17.0 software was used for statistical analysis. A chisquare test was used to compare different srtA sequences between the caries-active group and the caries-free group. A Pvalue below 0.05 was used to indicate a statistically significant
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difference between the two groups. BioEdit software was used to analyze the results of the sequencing.
3.
Results
3.1.
PCR products
A 941 bp DNA fragment carrying the srtA gene was amplified from the chromosome of all clinical bacterial strains by PCR. All 30 strains demonstrated clear banding patterns and presented a single positive band of 900–1000 bp in length.
3.2.
Sequencing results
1. The 741 bp srtA genes from the 30 clinical strains were sequenced. None of the srtA genes in the sequences trains had a base insertion or deletion.
2. Point mutations were present in the sequences of one of the clinical isolates (Fig. 1). In the caries-active group, 12 strains had point mutations, and the remaining three strains had no mutations for a total mutation percentage of 80%. In the caries-free group, all of the strains had point mutations for a total mutation percentage of 100%. There was no significance difference between the two groups (chi-square test, P = 0.224). 3. The sequencing revealed 20 mutation loci among the 30 clinical isolates. Half of the mutations were silent mutations. The remaining mutations were missense mutations. The transversion of the amino acids according to codon were shown in Table 1. 4. The silent mutation loci of the clinical isolates included position 78, 99, 150, 165, 186, 222, 249, 261, 312, and 636. The mutation loci details were listed in Table 2. 5. The missense mutation loci of the clinical isolates included positions 23, 34, 36, 47, 112, 114, 168, 176, 470, and 671. The mutation loci details are listed in Table 3. The mutation loci of 23, 34, 36, 47, and 470 were only noted in the caries-active group. Locus 168 was only noted in the caries-free group.
Fig. 1 – Point mutation in a clinical isolate (number is caries active 1), as indicated by srtA sequence. We can saw the point mutations occurred in the 78, 99, 112, 114, 222, 249, 470 locus base in the caries active 1 clinical isolate.
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Table 1 – Transversion of amino acid according to codons. Mutation
Base site
UA159
Clinical isolates
Codon
Amino acid
Codon
Amino acid
Silent mutation
78 99 150 165 186 222 249 261 312 636
GGT ACT TCT AAA GCT TCC GCA GCT AAC GCC
Glycin Threonine Serine Lysine Alanine Serine Alanine Alanine Asparagine Alanine
GGC ACC TCC AAG GCG TCT GCG GCC AAT GCT
Glycin Threonine Serine Lysine Alanine Serine Alanine Alanine Asparagine Alanine
Missense mutation
23 34 36 47 112 114 168 176 470 671
AGG AGT AGT ACC GCC GCC GAT CAC CGT AAT
Arginine Serine Serine Threonine Alanine Alanine Asparagine Histidine Arginine Asparagine
AAG GGC GGC ATC ACT ACT GAG CGC CAT ACT
Lysine Glycin Glycin Isoleucine Threonine Threonine Glutamic acid Arginine Histidine Threonine
Table 2 – Details of silent mutation sites in carie-active group and caries-free group. Clinical isolates
CA1a CA2 CA3 CA4 CA5 CA6 CA7 CA8 CA9 CA10 CA11 CA12 CA13 CA14 CA15 CF1c CF2 CF3 CF4 CF5 CF6 CF7 CF8 CF9 CF10 CF11 CF12 CF13 CF14 CF15 a b c
Base loci 78
99
T!Cb T!C T!C T!C T!C T!C
T!C T!C T!C T!C T!C T!C
T!C T!C
T!C
T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C T!C
T!C T!C T!C T!C T!C T!C
150
186
A!G A!G
T!C T!C T!C T!C T!C T!C
A!G A!G A!G A!G A!G A!G
T!C T!C T!C T!C T!C T!C T!C
A!G A!G A!G
T!C T!C T!C T!C T!C T!C
165
T!C
A!G A!G A!G A!G A!G A!G A!G
222
249
C!T C!T C!T C!T C!T C!T
A!G A!G A!G A!G A!G A!G
261
312
636
C!T C!T C!T C!T C!T
T!C T!C T!C T!C T!C T!C
T!G T!G T!G
T!C T!C T!C C!T C!T C!T C!T C!T C!T
A!G A!G A!G A!G A!G A!G
C!T C!T C!T C!T C!T C!T
C!T C!T C!T C!T
T!C T!C T!C C!T C!T C!T
A!G A!G A!G
CA, caries-active group. T!C, T represents the 78 locus base in UA159, C represents the 78 locus base in the clinical isolate. CF, caries-free group.
C!T C!T C!T
C!T
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Table 3 – Details of missense mutation sites in carie-active group and caries-free group. Clinical isolates
Base loci 23
34
36
47
a
CA1 CA2 CA3 CA4 CA5 CA6 CA7 CA8 CA9 CA10 CA11 CA12 CA13 CA14 CA15 CF1c CF2 CF3 CF4 CF5 CF6 CF7 CF8 CF9 CF10 CF11 CF12 CF13 CF14 CF15 Chi-square test a b c d
4.
112 G!A G!A G!A G!A G!A G!A
G!A G!A
A!G A!G
T!C T!C
C!T C!T
G!A
A!G
T!C
C!T
b
114
168
176
C!T C!T C!T C!T C!T C!T
470 G!A
A!C A!C A!C A!C A!C
A!G A!G A!G A!G A!G A!G
G!A
A!G A!G A!G
d
P = 0.224
P = 0.224
P = 0.224
P = 0.224
G!A G!A G!A G!A G!A G!A
C!T C!T C!T C!T C!T C!T
G!A G!A G!A P = 0.466
C!T C!T C!T P = 0.466
671
T!G T!G
T!G T!G
A!G A!G A!G A!G A!G A!G A!G
P = 0.100
P = 0.272
P = 0.483
A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C A!C P = 0.100
CA, caries-active group. G!A, G represents the 112 locus base in UA159, A represents the 112 locus base in the clinical isolates. CF, caries-free group. There is no significant statistical difference between the two groups regarding the mutation rate at each locus (P > 0.05).
Discussion
It has long been known that considerable phenotypic variation exists within S. mutans strains.18 This variation can be a consequence of a variety of different genetic events, including local point mutations, translocations and inversions. The availability of the full genome sequence of S. mutans UA159 has allowed comparison between strains at the genome level. The PCR-based double sequencing approach identifies single point mutations. Though restriction fragment length polymorphism (RFLP) is a classic method of biosystems gene structure analysis, it is not easy to select the proper restriction enzymes and the precise restriction enzyme cutting sites. In comparison to RFLP, the PCR-based double sequencing approach is considerably more objective in indicating the real genomic variations that may not always be visible via the electrophoretic band patterns in RFLP. Sortase has been shown to be a protease that catalyzes the cell wall anchoring of surface proteins containing an LPXTG motif in gram-positive bacteria. The srtA gene is required for cell wall anchoring and the surface display of antigen I/II (P1),5 glucan binding protein C (GbpC),19 dextranase,20 and surface proteins with LPXTG motif sorting signals. The inactivation of
srtA causes a decrease in biofilm formation and colonization.8,21 SrtA plays an important role in modulating the cariogenicity of S. mutans.5 The results of the present study indicated that clinical isolates of S. mutans had full-length srtA genes when compared to S. mutans UA159. All clinical isolates possessed point mutations. These results suggest those clinical strains have undergone genetic evolution. The two study groups have 20 mutation sites overall with both missense mutations and silent mutations represented. A missense mutation is a point mutation in which a single nucleotide change results in a codon that code for a different amino acid. A silent mutation is a DNA mutation that does not result in a change to the expressed amino acid. The structure of sortase reveals that the absolutely conserved His120, Cys184, and Arg197 residues of sortases reside within the active site.22 The 20 mutation sites identified in the present study were not in the sortase enzyme’s active sites. This finding means that the point mutations of the clinical strains did not affect the activity of the sortase enzyme. The missense mutation rate between the two groups was not significantly different. This probably reflects that the function of the srtA gene in clinical isolates is highly
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conserved. Similar results have been reported in other studies.23,24 On the other hand, the sample was relatively small for difference analysis between the two groups. The dmft 5 set as cut-off value probably narrow the selection objects of caries-active group. These two points may have some importance in the lack of significance. However, the results of the present study still provide some clues for the study of srtA polymorphisms in the future. The sequences were compared to UA159, which is a cariogenic strain of S. mutans. There were mutational sites observed in the cariesactive group that had no effect on cariogenicity, and mutation sites were observed in the caries-free group that modified cariogenicity. These sites needs further study. There was no significant difference in srtA enzyme activity between the two groups. These results indicate that the genetic diversity within the S. mutans srtA gene is not related to the severe caries experience in deciduous dentition. Further studies are needed to explore more fully the relationship between srtA and ECC.
5.
Conclusions
The results of the present study suggest that the genetic diversity within the srtA gene of S. mutans is not related to severe caries experiences in deciduous dentition. There were no detected genetic polymorphisms of the srtA gene in S. mutans isolates taken from two-year-old children suffering early childhood caries.
Acknowledgments Funding: This work was supported by the Guangdong Provincial Science & Technology project (Grant No. 2007B060401026). Competing interests: The authors declare that there are no conflicts of interests. Ethical approval: Ethical Approval has been given by the Research Ethics Committee of Guanghua School of Stomatology, Sun Yat-sen University (No: 2009-01).
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